Characterizing Changes in Oral Microbiota With Cardiometabolic Risk Factors The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation WARSI, AHMED IBRAHIM. 2020. Characterizing Changes in Oral Microbiota With Cardiometabolic Risk Factors. Master's thesis, Harvard Medical School. Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365240 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Characterizing Changes in Oral Microbiota with Cardiometabolic Risk Factors by Ahmed Ibrahim Warsi A Dissertation Submitted to the Faculty of Harvard Medical School in Partial Fulfillment of the Requirements for the Degree of Master of Medical Sciences in Clinical Investigation (MMSCI) Harvard University Boston, Massachusetts April 2020 Area of Concentration: Oral Microbiology, Oral-Systemic Health Connection Project Advisor: Dr. Jo. Max Goodson I have reviewed this thesis. It represents work done by the author under my guidance/supervision. Primary Mentor: Dr. Tsute Chen Page 1 of 116 Table of Contents Acknowledgments...…………………………………………………………………………...……………...…………03 Thesis Committee Members..………………………………………………………………………..………………04 Overview……………………………………………………………………………………………………..………………05 Project I: Changes in Oral Microbiota with the Development of Adolescent Obesity, and Effect-Modification with Metabolic and Inflammatory factors……...…………...……………………12 Abstract…………………………………………………………………………………………………….…………………14 Introduction……………………………………………………………………………………………..….………………15 Methods………………………………………………………………………………………………….…………...………16 Results…………………………………………………………………………………...……………….…………...………20 Discussion………………………………………………………………………………………………….…………...……25 References…………………………………..………………………………………………………….…………...……….30 Figures………………………..……….………………………………………………………………….…………...………37 Tables………………………………….………………………………………………………………….…………...………40 Supplement…….…………………………..………………………………………………………….……….…...……….49 Project II: Reduction of Nitrate and Nitrite-reducing Oral Microbiota is Associated with Pediatric Hypertension……………………..……………………………………………………….………………….67 Abstract…………………………………………………………………………………………………….………………….69 Introduction……………………………………………………………………………………………..….……………….70 Methods………………………………………………………………………………………………….…………...……….72 Results…………………………………………………………………………………...……………….…………...……….76 Discussion………………………………………………………………………………………………….…………...……79 References…………………………………..………………………………………………………….…………...……….88 Tables………………………………….………………………………………………………………….…………...……….98 Figures………………………..……….………………………………………………………………….…………...…….103 Supplement…….…………………………..………………………………………………………….…………...……...104 Summary of Projects I and II…………...…………...………………………..………………….…………...…….112 Discussion and Perspectives of Projects I and II…....………………..………………….…………...…….114 Page 2 of 116 Acknowledgments I would like to acknowledge and thank our truly amazing Program Directors – Dr. Ajay Singh, and Dr. Finnian Mc Causland; Program Managers - Katie Cacioppo, Katie King, and Claire Francis; and all MMSCI faculty and colleagues, who were instrumental in teaching me clinical research skills and helping me graduate with a Masters’ degree in Clinical Investigation. Further, I would like to commend the efforts of our Program Directors who created a positive atmosphere in our cohort, which made this intense and challenging journey, easy for us to sail through. I would also like to thank my Forsyth mentors, Dr. Tsute Chen, and Dr. J. Max Goodson, who believed in me, trained me to the best of their abilities and polished my skills in clinical and translational research. Their dedication and hard work helped me push my boundaries to truly understand the field of microbiome research. I would like to thank my MMSCI Program representative, Dr. Enid Martinez, who helped me in shaping a concise research question from the humungous multi-omics dataset that I have been working on. I would also like to thank Dr. Bruce Paster (external thesis reviewer) who reviewed my thesis and gave instrumental feedback. I would also like to thank Dr. Hatice Hasturk, Dr. Ana Colombo, and Prasad for their immense help in dealing with oral microbiome data. Last, I would like to acknowledge my mom, dad, brother, grandparents, friends, and my extended family, because without their prayers, moral, and financial support I would not have been here. I would like to dedicate this work to my uncle, who recently passed away due to COVID-19. He partly sponsored my tuition at Harvard, and without his support, this journey would not have been possible. Page 3 of 116 Thesis Committee Members: 1. Student: Dr. Ahmed Ibrahim Warsi 2. Primary Mentor: Dr. Tsute Chen 3. Co-Primary Mentor: Dr. J. Max Goodson 4. External Content Advisor: Dr. Bruce Paster 5. MMSCI Program Representative: Dr. Enid Martinez 6. Program Director: Dr. Ajay Singh 7. Program Co-Director: Dr. Finnian Mc Causland Page 4 of 116 Overview Cardiometabolic risk factors (CMR) such as obesity and hypertension in adolescents are independent risk predictors for the development of cardiovascular disease in adulthood (1,2). Recent evidence has demonstrated the dysbiosis of oral and gut microbiota with CMR (3–5). While longitudinal studies examining the association between oral dysbiosis with the evolution of CMR in children are lacking. I aim to examine changes in oral microbiota with the development of obesity (Project-I), and hypertension (Project-II), among adolescents in Kuwait Healthy Lifestyle Study Cohort (KHLS). The Human Microbiome Project indicated that the oral and gut microbiome overlap in nearly 45% of the population (6). The premise that oral and gut have an anatomical connection and that ingesting oral bacteria could be a source for colonization of gut microbiota is plausible (7,8). In inflammatory systemic diseases, increased numbers of oral bacteria have been observed in the intestine (9–11). Moreover, it has been reported that an increased abundance of pathogenic, gram-negative, oral Bacteroidetes, such as Porphyromonas gingivalis can alter the gut microbial ecosystem (12). Numerous studies have indicated that obesity-associated gut microbiome has increased capacity for energy harvest,(13–15) by digesting otherwise indigestible complex polysaccharides, the gut microbiota is now recognized as an additional contributing factor towards the pathophysiology of obesity (13,16). However, it is still not clear where did the gut microbiota come from? And how could oral microbiota influence the gut microbiome in the development of obesity (8)? To bridge the gap, we compared the oral microbiota in healthy, overweight, and obese phenotypes. To place the oral dysbiosis in context with the Page 5 of 116 pathophysiology of obesity, I compared the study results with existing studies on oral and gut dysbiosis and obesity development. Project-I: The KHLS cohort study was envisioned following the observation published in 2009 (17), suggesting that obesity could be the result of oral bacterial infection. The KHLS investigators reasoned that by the selection of children from a population with high levels of adult obesity, we would be able to determine if obesity could be predicted. In this longitudinal study, I analyzed data from 67 adolescents, sampled at visits 1 and 2, two- years apart. In this cohort, 28 healthy, 33 overweight, and 6 obese children at baseline were followed for two-years. At visit-2, 19 healthy, 10 overweight, and 38 obese subjects were identified. In summary, 47% of our baseline subjects became obese by visit 2. These remarkable changes in host-phenotypes concerning weight gain, allowed me to characterize changes in oral microbiota with changes in host phenotypes – using both cross-sectional and longitudinal analyses. I also observed drastic changes in systemic inflammatory and metabolic factors, which could also influence changes in oral microbiota (18). To compare oral microbial changes across healthy, overweight, and obese phenotypes; and to investigate the possibility of effect-modification with inflammatory and metabolic factors, I constructed three sub-cohorts comparing: (i) healthy-vs-overweight subjects, (ii) overweight-vs-obese subjects, and (iii) healthy-vs-obese subjects. Project-II: From the analysis, I learned that Kuwait has one of the highest prevalence of adolescent hypertension (nearly 40% within KHLS cohort), as compared to 5.5% reported for the USA (19). This raises concerns about the involvement of multiple pathways responsible for a higher prevalence of cardio-metabolic risk factors amongst the KHLS Page 6 of 116 cohort. Besides other known cardiometabolic risk factors that have already been reported (20), I assessed the role of the “Enterosalivary-Nitrate-Nitrite-NO pathway” with pediatric hypertension (21,22). The second project was aimed at assessing whether the changes in nitrate and nitrite-reducing oral bacteria, could influence changes in blood pressure amongst the KHLS cohort. The enterosalivary nitrate-nitrite-nitric oxide pathway is an alternative route of nitric oxide generation, potentially linking the oral microbiome to blood pressure regulation (21–25). I hypothesized that the reduction of nitrate and nitrite- reducing oral bacteria would be associated with an increase in blood pressure and higher odds of having hypertension. In both projects,